Campylobacter jejuni is a food-borne pathogen in humans that causes diarrhoeal disease. This enteric pathogen has evolved several antimicrobial resistance mechanisms and is recognized as a serious antibiotic resistance threat. One of the mechanisms conferring resistance to clinically relevant antibiotics is multidrug-resistance efflux pumps, which actively export drugs from the bacterial cell, such as the ubiquitous resistance-nodulation-cell division (RND) superfamily. These efflux pumps comprise an inner membrane protein, a periplasmic fusion protein and an outer membrane channel, and in the RND of C. jejuni (CmeABC) all three of those components are post-translationally modified with N-linked glycans. Although impairment of glycosylation pathways in bacteria has been shown to affect virulence, colonization and efflux pump activity, the direct role of this modification is elusive. In this study, Wren and colleagues directly link N-linked glycans to the activity of multidrug efflux pumps and thus antimicrobial resistance.
First, the authors report that a glycosylation-impaired C. jejuni strain exhibits decreased efflux pump efficiency, as measured by the accumulation of ethidium bromide. In addition, abrogation of CmeABC glycosylation also led to the increased susceptibility to four structurally different antibiotics compared to the wild type. These findings suggest that N-linked glycans have a direct effect on efflux pump activity and antibiotic resistance.
The authors went on to investigate how N-linked glycans modulate the function of CmeABC. To this end, they heterologously expressed nonglycosylated and glycosylated forms of the periplasmic fusion protein CmeA in Escherichia coli. Using circular dichroism spectroscopy, they found that N-linked glycans affect the global protein conformation of CmeA. Moreover, they showed that N-glycans enhance protein thermostability, and they suggest that this is due to slowing of the unfolding rate. In addition, glycosylation was shown to enhance the interaction between CmeA and CmeC, which is the outer membrane-associated channel of the tripartite efflux pump, which implies that glycosylation promotes the assembly of the multidrug efflux pump.
“N-linked glycans have a direct effect on efflux pump activity”
In summary, the study provides strong evidence that N-linked glycans in RND mediate antimicrobial resistance by enhancing the activity of multidrug efflux pumps. In particular, N-linked glycans slow protein unfolding, enhance thermostability and modulate protein–protein interactions, and may present a potential antimicrobial drug target.
Abouelhadid, S. et al. Characterization of posttranslationally modified multidrug efflux pumps reveals an unexpected link between glycosylation and antimicrobial resistance. mBio https://doi.org/10.1128/mBio.02604-20 (2020)
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Du Toit, A. Aiming at the sweet spot of resistance. Nat Rev Microbiol 19, 74–75 (2021). https://doi.org/10.1038/s41579-020-00494-4